FIJI/Image J for Quantification Hands on session

FIJI/Image J for Quantification Hands on session Dr Paul McMillan Biological Optical Microscopy Platform

Hands on demonstrations FIJI set up Line Profile Thresholding Area of stain Cell confluence Nuclei counting Cell segmentation Advanced segmentation Intensity over time Euclidean Distance Measurements Kymogram

FIJI set up Edit/Options/Colours Foreground: White Background: Black Selection: Yellow Process/Binary/Options Iterations: 1, Count: 1 Black Background (Tick ) Pad edges when eroding (Tick) EDM output: Overwrite Edit/Options/Memory and threads Set memory 60-70% of memory Plugins/Utilities/Monitor Memory Clear memory as we go Plugins/Utilities/Find commands Control L (PC) or Command L (Mac)

Line analysis Open Gams.tif, Gams (green) & Gams (red) Change line tool to segmented line (right click to change) Draw line along filament Restore selection (Control + Shift E) Plot line profile on gams (green) & gams (red) Analyse/plot profile, Control K (PC) or Command K (Mac) Save plot as xls Edit/Selection/Straighten, line width 10 pixels Change line colour (Edit/Options/Colours/Selection) Change width of line (Edit/Options/Line width) Save line as overlay (Image/Overlay/Selection) Control B (PC) or Command B (Mac)

Thresholding Open kidney (green).tif Image/adjust/auto threshold Select white objects on black background Show threshold values in log window Select one that s works best Find threshold values in log window Apply selected Threshold Image/Adjust/Threshold Control + Shift T (PC) Command + Shift T (Mac)

Area of stain Open kidney (green).tif Images/adjust/threshold Use method & settings identified in auto threshold Analyse/set measurements Area, integrated density, mean gray value, area fraction Limit to threshold Measure Analyse/measure Control M (PC) Command M (Mac) Right click (or Analyse/Set measurements) to change measurement settings

Cell confluency Open BPAE (green) Set threshold Image/Adjust/Threshold Control + Shift T (PC) Command + Shift T (Mac) Measure Analyse/Measure Control M (PC) Command M (Mac) Right click (or Analyse/Set measurements) to change measurement settings

Nuclei Counting Open BPAE (blue).tif Image/Adjust/Threshold Set lower threshold level = 70 Set higher threshold level = 255 Apply threshold Process Binary/watershed Analyze/analyze particles Size = 100-infinity Circularity = 0-1.0 Show = outlines Exclude on edges Tick Display results, Summarise, Exclude on edges

Cell segmentation

Cell segmentation Open Cell.tif (from Segmentation) & Duplicate Mask 1: Watershed Process/Find Maxima (Noise = 400, exclude on edges, segmented particles) Mask 2: Whole cell stain Threshold duplicated image (min = 388, max 4095) but don t apply yet Process/smooth & apply threshold Mask 3: Cell outlines Process/Image Calculator Mask 1 AND Mask 2 Analyze/Analyze Particles (Size = 250 Inf, exclude on edges, show masks) Invert LUT Process/Binary/Fill Holes Analyse the images Analyze/Set Measurement (Area, Shape, Int Den, Mean, Perimeter, Ferets, Display label. REDIRECT to Cell.tif Analyse Particles (size = 250-infinity, Show = outlines, display, clear, summarize, exclude on edges) Try as above, but also select ROI manager

Advanced segmentation Cytoplasmic masks (Cells minus nucleus) Open Nuclei.tif & Threshold (Save as Mask N) Process/Image calculator/ Mask 3 SUBSTRACT MASK N Perinuclear mask Open Mask N & Duplicate Process/Binary/Dilate (on Mask N-1.tif) & Process/Binary/Watershed Process/Binary/Erode (on Mask N.tif) Process/Image calculator/mask N-1 SUBTRACT Mask N

Intensity over time Open Calcium flux.tif Draw ROI on bottom right cell Analyze/Set Measurement Mean Gray value Display label Image/Stacks/Plot Z axis Profile Repeat on background Analyze/Tools/ROI manager Add multiple ROIs Show all Select More, Multi Measure, Measure all 50 slides, one row per slice

Euclidean distance measurement Open Nuclei.tif, apply threshold & create binary image Process/Binary/Options Configure EDM to 16 bit Edit/Invert Process/Binary/Distance Map Apply 16 colours LUT Analyze/Set Measurements Mean gray value, limit to threshold, display label Threshold to select background Analyze/Measure Average distance = 8.098 Pixels (read out is always in pixels) Calculate distance in microns on calibrated images Image/Properties, Control + Shift P (PC), Command + Shift P (Mac) Covert using pixel dimensions

Kymogram Open tracking.tif from Kymogram Image/Colour/Split channels Draw line form ROI tools (Hold shift to keep it straight) Select channel two and restore selection (Ctrl + Shift E) Select channel 1, Analyze/Multi Kymporaph/Multi Kymograph Set line width to 29 (must be an odd number) Image/Rename, rename channel 1 Repeat Kymograph (as above) for Channel 2 Merge Channels (Channel 1 = green, Channel 2 = Red)